With the completion of human genome project, there is now a focus on developing new DNA sequencing technology that will reduce the cost of sequencing dramatically without sacrificing accuracy, which will ultimately enable personalized medicine in healthcare (1). Current state-of-the-art DNA sequencing technology faces limitation in terms of cost, read-length, and throughput. In this regard, DNA sequencing by synthesis (SBS), where the identity of each nucleotide is detected immediately after its incorporation into a growing strand of DNA in a polymerase reaction, offers an alternative approach to address some of these limitations. An important requirement for the SBS approach is a 3′-OH capped fluorescent nucleotide that can act as a reversible terminator (2), where after the identification of the nucleotide incorporated in a DNA polymerase reaction, the 3′-OH capping group along with fluorescent label are removed to regenerate a free 3′-OH group thus allowing DNA chain elongation. The importance of removing the fluorescent label after each base identification is to make sure that the residual fluorescence from the previous nucleotide incorporation does not affect the identification of the next incorporated fluorescent nucleotide.
The speed and sequence read length of SBS depend on the yield of the cleavage efficiency of the fluorophore and the allyl group. Due to multiple steps required in the identification, removal of fluorescent label, and regeneration of 3′-OH group after each nucleotide incorporation in SBS, the loss of even a minor efficiency at each step may lead to inhibition of prolonged read length. For this reason, any improvement in efficiency within each cycle of nucleotide identification, fluorophore removal, and 3′-OH regeneration can have significant impact on read length, thus tackling the physical limits in DNA sequencing by synthesis.